F2MutantScreen-non"clonal"

1.Mutagenizemixedpopulationwormsaspernormal.Besttodothisfirstthinginthemorninginordertogetseverallaysthenextday.

2.Putthewholemixtureonplatesandletrecover10minutes(Erikwaits2hours).

3.PickL4animals,putmanyperplateonnewplates.Agetheanimals24hours,atwhichpointtheywillbeearlygravidadults.(Note:byaging24hours,youavoidthefirsteggsthatthemutagenizedanimalslay.Thesemayhavealreadybeenthroughmeiosisatthetimeofmutagenesis,sothatmutationsmaynothavebeenfixedonbothstrandsoftheDNA.BynottakingthefirstProgeny,youavoidthistheoreticalconcern.Thereisalsoaconcernabouttakingtheverylastprogeny,fromthirddayadults.Here,youmaygetjackpots,asthemutagenizedcellsmayhaveundergoneseveralroundsofmitosisbeforemeiosis).

4.Put3-4gravidadultP0perplateon209cmplates.

5.MovetheP0tonewplatesevery3-4hours;thisshouldgiveyou~50-80F1eggsperplate.Dothisallday,giving60-80totalplates.Note:50-80F1onaplatewillallowyoutogetfirstdayadultF2sontheplatesbeforetheplatesstarveout.Toget2nddayadults,needtogetlessthan~50F1onaplate.

AnalternativeIpreferistonotuseseriallays;lotsofanimalsareEMStreated,agedexactly24hoursaftertheendoftheEMStreatment,andasinglelayistaken.Thisseemstobemoreconvenient.Numbers:Using3F1perplateanda3hourlay,thefirstlay(starting24hoursaftertheendoftheEMStreatment)willgive~35F1perplate.Using4F1perplate,4hourlay,starting24hourspostEMS,get60-80,average70,F1perplate.ThiswillgivegravidfirstdayF2adultswithF3eggs,andthenstarvesoonthereafter,assumingyoustartwithathicklargelawn.

6.Erik"sway:Putonebatchof20platesat25°.Puttherestat15°todelaytheirdevelopment,givingyoubreathingroomforthescoring.Movethenextsetupto25°after1.5days,andthethirdsetto25°onedaylater.

6.5Michael"sway:Putonebatchat20°,andtwootherbatchesat15°.Aftertwodays,movea15°batchto20°.Afteranothertwodays,movethelastbatchto20°.Thiswillspacethebatchesoutsothattheywillbescored3daysinarow.Thelastplatewillbescoredonthe8thdayaftertheEMStreatment.

7.Monitoreachsetofplates.WhentheveryfirstF2L1"sbegintohatch,counttheF1"sonsomeoralltheplates(dependingonhowuniformtheplatesare)toestimatethenumberofgenomesscreened.(BethSawinwaitsuntilthereare3-foldembryosandmaybeL1"shavebarelyhatched).EachF1represents2haploidmutagenizedgenomesscreened.ThenremovetheF1fromtheplateswhileleavingtheF2eggsontheplatetodevelopandlaterbescored.ThisisaccomplishedbydumpingsomeM9orSbasalgentlyoneachplate(useagenerousamountthatalmostfillstheplatetothetop),swirlgentlyfor1-2seconds,andpouroff.(Ifthereisapatchofcontaminationontheplate,cutitofffirstsoasnottospreadit).Dothistoeachoftheplates,andsetthemasiderightsideup.TherewillbeafewF1adultsremainingontheplate,whichwillcrawlbackonthelawnbecomingeasilyvisIBLeafteracoupleofminutes.Toremovethese,hookupapasteurPipettetoavacuumlineandshoveasterileyellowtipontheend.Underadissectingscope,usingthe12Xobjective,scanforandsuckofftheremainingworms.Becarefulnottodamagetheagarsufacehereorthewormsmayburrowintotheplate.

8.Scoretheanimalsattheappropriatetime.Iflateradultsaretobescored(e.g.fortheEglphenotype),itmaybenecessarytomodifythisprotocolforshorterlaysinordertokeeptheplatesfromgoingstarved.

9.Expectabout4-5twitchers(unc-22mutants)perplateifthingsaregoingwell.Willgetafewsiblingsofeachphenotypeperplate.Youcanonlykeeponeanimalperplateifyouwanttoknowthatyourmutantsareindependent.However,pickseveralanimalsperplate,sinceabout1/3oftheanimalswillbesterileperhapsduetoothermutations.ItisErik"simpressionthattheveryfirstlaydoesn"tgiveasmanymutantsasthenextcouple;perhapstheveryfirstgermcellstomaturearen"tEMSmutagenizedaswellasearlierstagegermcells.